Chemiluminescent reagent for detecting alkaline phosphatase

Abstract

Described herein are 1, 2-dioxetane useful as chemiluminescent probes, diagnostic agents, and imaging agents. Compositions comprising such compounds and methods of use thereof are also described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 369,879, filed on July 29, 2022, which is incorporated by reference as if fully set forth herein. Background Art

[0003] Chemiluminescent dioxetanes are strained cyclic peroxides that can undergo rapid decomposition to produce an excited transient species that subsequently decays to the ground state by light emission.

[0004] Such compounds can be used as luminescent probes in a range of assays, including enzyme activity assays, immunoassays, and DNA detection assays. Chemiluminescence-based assays can provide excellent sensitivity because, unlike fluorescence- and absorption-based assays, they do not require light excitation.

[0005] Dioxetanes can be generated in situ at the time of their use or pre-prepared in a stable form and subsequently activated. When generated in situ by oxidation of a precursor olefin, chemiluminescent dioxetanes can also be used as a detection or imaging method for reactive oxygen species (ROS). Some examples of stable chemiluminescent dioxetanes are 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxetane-3,2'-adamantane]. This compound, also known as PPD can be activated when treated with alkaline phosphatase (ALP). ALP is an enzyme that catalyzes the hydrolysis of phosphate groups. Once activated, the resulting compound then undergoes 1,2-dihydropyridine hydrolysis. The alkyl ring fragments and emits light, thus being used as a luminescent probe in the ALP labeling assay.

[0006] Dioxetane compounds have been developed that emit sensitively and strongly under non-aqueous conditions. However, such compounds emit weakly in aqueous media and may take a long time to reach maximum luminescence after contact with the desired analyte. Summary of the invention

[0007] There is a need for dioxetanes that provide a rapid response to the presence of an analyte and the concentration of the analyte or both. There is also a need for dioxetanes that are highly emissive and suitable for use in aqueous environments. The present disclosure describes compositions comprising a combination of dioxetanes and one or more selected surfactants that provide unexpectedly improved emission results in aqueous environments. For example, certain combinations can provide significant relative differences (more than 20 times higher) in brightness in relative light units (RLU), depending on the combination of the dioxetanes and the surfactants. A significant benefit of the compositions described herein is that the detection limit of alkaline phosphatase can be improved. For example, using the compositions described herein, 1×10 -12 mol / μL to 1×10 -23 For example, in some aspects, the compositions described herein can be used to detect ALP in a composition having an ALP concentration of at least 1×10 -23 mol / μL, at least 1×10 -22 mol / μL, at least 1×10 -21 mol / μL, at least 1×10 -20 mol / μL or at least 1×10 -17 mol / μL; In addition or alternatively, the composition described herein can be used to detect ALP in a composition, wherein the ALP concentration of the composition is at most (i.e., at most) 1×10 -21 mol / μL, up to 1×10 - 20 mol / μL, up to 1×10 -19 mol / μL, up to 1×10 -18 mol / μL, up to 1×10 -15 mol / μL, up to 1×10 -10 mol / μL or up to 1×10 -5 mol / μL. In some exemplary aspects, the compositions described herein can be used to detect: AP4, whose ALP concentration is 1.2×10 -15 mol / μL; AP6, whose ALP concentration is 1.2×10 -17 mol / μL; AP8, its ALP concentration is 1.2×10 -19 mol / μL; and AP9, whose ALP concentration was 1.2×10 -20For example, using the compositions described herein, one can, for example, in a composition comprising 5 μL to 50 μL (e.g., 10 μL AP9) of an alkaline phosphatase composition diluted in 50 μL to 250 μL (e.g., 100 μL) of a buffer (e.g., comprising 1×10 -11 mol to 1×10 -22 mol alkaline phosphatase), 1×10 -11 mol to 1×10 -22 mol alkaline phosphatase (e.g. 1×10 -14 mol, 1×10 -15 mol, 1×10 -16 mol, 1×10 -17 mol, 1×10 -18 mol, 1×10 -19 mol, 1×10 -20 mol, 1×10 -21 mol, 1×10 -22 mol, 1×10 -23 mol; 1×10 -13 mol to 1×10 -22 mol, 1×10 -11 mol to 1×10 -20 mol, 1×10 -13 mol to 1×10 -18 mol, 1×10 -15 mol to 1×10 20 , or 1×10 -14 mol to 1×10 -17 mol alkaline phosphatase).

[0008] The present disclosure provides compositions comprising a compound of formula I and its salts, and at least one surfactant:

[0009]

[0010] Where R 1 and R 2 Each is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon to which they are attached, they provide C5-C 10 Cycloalkyl ring;

[0011] R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl;

[0012] R 4It is C2-C 10 alkenyl;

[0013] R 5 Is H or C1-C 10 alkyl;

[0014] X is a phosphate ester.

[0015] The terms "surfactant" and "enhancer" are used interchangeably herein. Some examples of such surfactants include, but are not limited to Surfactants, which are also referred to herein interchangeably as Surfactants include polymers Surfactants and small molecules Surfactants. Polymerization Some examples of surfactants include Pendant polyethylene type polymers as disclosed in US Pat. No. 5,393,469.

[0016] Exemplary Aggregation Enhancers include polyvinylbenzyltributyl chloride Polyvinylbenzyl trioctyl chloride and polyvinylbenzyltributyl chloride A copolymer having two quaternary ammonium or dicationic compounds of the group, such as those disclosed in U.S. Pat. No. 5,451,347.

[0017] polymerization Some examples of reinforcing agents include polymers comprising repeating units (A), repeating units (B), or both:

[0018]

[0019] Wherein "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl". The molar ratio of repeating unit (A) to repeating unit (B) can be in the range of 1:100 to 100:1 (e.g., 100:1 to 1:1, including, for example, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 70:30, 75:25, 60:40 or 50:50). Weight average molecular weight (MW) w) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M w In some embodiments, M may be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. w In some embodiments, M w In some embodiments, M w In some embodiments, M w In some embodiments, M w The copolymer may be a random copolymer, a block copolymer or a random block copolymer.

[0020] polymerization Some examples of reinforcing agents include polymers of the following formula:

[0021]

[0022] wherein m and n are each independently an integer from 0 to 1000 (e.g., 1 to 500, 250 to 1000, 300 to 900, 50 to 500, or 250 to 750). The ratio of m:n can be, for example, from 100:1 to 1:1, including, for example, a ratio of m:n of 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 70:30, 75:25, 60:40, or 50:50; m+n is in the range of 100 to 1000 (e.g., m+n can be 100 to 600, 200 to 500, or 300 to 600); the weight average molecular weight (M w ) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M wIn some embodiments, M may be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. w In some embodiments, M w In some embodiments, M w In some embodiments, M w In some embodiments, M w The copolymer may be a random copolymer, a block copolymer or a random block copolymer.

[0023] Exemplary Aggregation Enhancers / surfactants also include polymers containing the following structure Surfactants:

[0024]

[0025] wherein z is an integer from 2 to 1000 (e.g., from 2 to 500, from 100 to 600, from 450 to 800, from 300 to 600, from 2 to 250, from 50 to 250, from 100 to 300, from 150 to 400, from 175 to 400, from 50 to 300, from 100 to 500, from 150 to 400, or from 10 to 200). The weight average molecular weight (M w ) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M w In some embodiments, M may be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. w is 70,000, 80,000, 100,000 or 220,000 g / mol. In some embodiments, Mw In some embodiments, M w In some embodiments, M w In some embodiments, M w is 300,000 g / mol. Some examples of enhancers / surfactants include those comprising the following structure:

[0026] and / or

[0027] wherein z is an integer from 2 to 1000 (e.g., from 2 to 500, from 100 to 600, from 450 to 800, from 300 to 600, from 2 to 250, from 50 to 250, from 100 to 300, from 150 to 400, from 175 to 400, from 50 to 300, from 100 to 500, from 150 to 400, or from 10 to 200). The weight average molecular weight (M w ) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M w In some embodiments, M may be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. w is 70,000, 80,000, 100,000 or 220,000 g / mol. In some embodiments, M w In some embodiments, M w In some embodiments, M w In some embodiments, M w It is 300,000g / mol.

[0028] Exemplary Small Molecules Enhancers / surfactants include small molecule surfactants of the formula:

[0029]

[0030] in:

[0031] R 12 To R 14 Each is independently C1-C 10 alkyl;

[0032] R 15 is arylalkyl; and

[0033] X - is a counterion (e.g. chloride). 15 It may be an arylalkyl group substituted with a C2-C8 alkenyl group or a group of the formula:

[0034] where k is 0, 1 or 2; R 16 To R 18 Each is independently C1-C 16 Alkyl. 16 To R 18 Can be C4-C independently 10 Alkyl. C2-C8 alkenyl can be a group of the formula:

[0035] Where R 19 It is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl.

[0036] Some examples of surfactants contemplated for use with the dioxetane compounds described herein also include fluorescein surfactants (also interchangeably referred to herein as "fluorescein enhancers"), such as those having Formula V / VI:

[0037] It is usually balanced with:

[0038]

[0039] Where R 6 It is C6-C 20 Alkyl, such as C6-C 15 、C8-C 16 , C 10 -C 14 and C 12 -C 20Alkyl. Some examples of compounds of formula V / VI include 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein, 5-stearoylaminofluorescein, etc. The present disclosure also provides aqueous compositions comprising one or more chemiluminescent dioxetane compounds having a peak RLU greater than 8,000 (e.g., at 37° C.) and / or an emission half-life (T 1 / 2 ) is 3 minutes or less (e.g., at 37°C).

[0040] Combinations of any of the above surfactants / enhancers (e.g., two or more small molecules polymerization Combinations of surfactants / enhancers and fluorescein are also contemplated herein. However, the use of only one surfactant is also contemplated herein.

[0041] The present disclosure also provides methods for determining at least one of the presence and concentration of an analyte (e.g., ALP or ALP conjugated to an analyte of interest) in a sample, comprising contacting the sample with a compound of Formula I and monitoring luminescence of the sample.

[0042] Several advantages, some of which are unexpected, are achieved through various embodiments of the present disclosure. For example, when the compounds of Formula I were first developed, it was believed that for bright chemiluminescent probes, R 4 The electron-withdrawing group at the R position is necessary. See, for example, ACS Cent. Sci. 2017, 3, 349-358. Based on this knowledge, one skilled in the art would not expect that the compounds of Formula I would be effective, bright chemiluminescent probes because they have 4 In contrast, the compound of formula I lacks an electron-withdrawing group at R 4 An electron-donating vinyl group is present at the position.

[0043] Various compounds and compositions described herein can advantageously provide fast, high-intensity luminescent signals in non-aqueous media, aqueous media or both. Significant advantage is that the determination involving such compounds or compositions can be carried out faster than those of compounds with the characteristics of the compounds and compositions lacking the current description, because the maximum luminescent signal is obtained faster. In addition, the compounds and compositions of the present disclosure provide the luminescent intensity of improvement, including in aqueous media. The compounds and compositions of the present disclosure also provide the signal-to-noise ratio of improvement and the background signal of reduction. Due to such favorable characteristics, multiple embodiments of the present disclosure can provide methods or test kits, which can detect the analyte in aqueous or non-aqueous samples in less than 3 minutes, less than 1 minute, less than 30 seconds or less than 15 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a graph of relative luminescence units as a function of time (minutes) for compounds "VPPD," "VMPD," and "PPD" using the fluorescein surfactant used in LP530. The fluorescein surfactant used in LP530 comprises a fluorescein surfactant of Formula V / VI and cetyltrimethylammonium bromide (CTAB). "VPPD," "VMPD," and "PPD" have structures further described herein.

[0045] Figure 2A is a graph of relative luminescence units as a function of time (minutes) for compounds "VPPD," "VMPD," and "PPD" using "EXL surfactant," wherein the EXL surfactant has a structure further described herein.

[0046] Figure 2B is a graph of RLU as a function of time at various "VPPD" concentrations (ranging from 50 mg / L to 500 mg / L) while keeping the EXL concentration constant (ranging from 100 mg / L to 250 mg / L).

[0047] Figure 2C is a graph of RLU as a function of time at various "EXL Surfactant" concentrations (ranging from 100 mg / L to 500 mg / L) while holding the VPPD concentration constant (ranging from 50 mg / L to 500 mg / L).

[0048] Figure 3 Graphs of relative luminescence units of compounds "VPPD," "VMPD," and "PPD" in EXL surfactant, or "VPPD" in fluorescein surfactant for LP530, "VMPD" in fluorescein surfactant for LP530, or LP530 (i.e., PPD in fluorescein surfactant) as a function of time (minutes), normalized to the intensity of VPPD in EXL surfactant.

[0049] Figure 4 It is a variety of A graph of the relative luminescence units of VPPD in surfactant as a function of time (minutes); also shown are the relative luminescence units of LP530 as a function of time (minutes).

[0050] Figure 5 Is Aggregation Plot of the RLU of "VPPD" in surfactant as a function of molar ALP.

[0051] Figure 6is a graph of the RLU of "VPPD" in "EXL Surfactant" and the RLU of LP530 as a function of time (minutes). In this experiment, VPPD and polymer 100 μL of the enhancer (EXL) solution was mixed with 10 μL AP10 (1.2×10 -21 100 μL LP530 solution was mixed with 10 μL AP8 (1.2×10 -19 mol / μL ALP) were mixed and incubated at 37°C. It is worth noting that the ALP concentration of AP8 is 100 times higher than that of AP10. In other words, a 100-fold higher ALP concentration is required under LP530 to obtain almost the same light signal as that achieved under VPPD and EXL.

[0052] Figure 7 is a graph of RLU of VPPD EXL and LP530 background as a function of time (minutes), where "background" refers to the signal obtained for a VPPD composition comprising EXL or LP530, each without any ALP. The signal is generated by thermal decomposition of dioxetane when incubated at 37°C. DETAILED DESCRIPTION

[0053] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplary subject matter is not intended to limit the claims to the disclosed subject matter.

[0054] The compounds of the present disclosure can be used in chemiluminescent applications, such as alkaline phosphatase (ALP) assays and chemical probes. A significant benefit of the compositions described herein is that the ALP detection limit can be improved. For example, using the compositions described herein, 1×10 -12 mol / μL to 1×10 -23 For example, in some aspects, the compositions described herein can be used to detect ALP in a composition having an ALP concentration of at least 1×10 -23 mol / μL, at least 1×10 -22 mol / μL, at least 1×10 -21 mol / μL, at least 1×10 -20 mol / μL or at least 1×10 - 17 mol / μL; as a supplement or alternative, the composition described herein can be used to detect ALP in a composition having an ALP concentration of at most 1×10 -21mol / μL, up to 1×10 -20 mol / μL, up to 1×10 -19 mol / μL, up to 1×10 - 18 mol / μL, up to 1×10 -15 mol / μL, up to 1×10 -10 mol / μL or up to 1×10 -5 mol / μL. In some exemplary aspects, the compositions described herein can be used to detect: AP4, whose ALP concentration is 1.2×10 -15 mol / μL; AP6, whose ALP concentration is 1.2×10 -17 mol / μL; AP8, its ALP concentration is 1.2×10 -19 mol / μL; and AP9, whose ALP concentration was 1.2×10 - 20 For example, using the compositions described herein, one can, for example, in a composition comprising 5 μL to 50 μL (e.g., 10 μL AP9) of an alkaline phosphatase composition diluted in 50 μL to 250 μL (e.g., 100 μL) of a buffer (e.g., comprising 1×10 -11 mol to 1×10 -22 1×10 -11 mol to 1×10 -22 mol alkaline phosphatase (e.g. 1×10 -14 mol, 1×10 -15 mol, 1×10 -16 mol, 1×10 -17 mol, 1×10 -18 mol, 1×10 -19 mol, 1×10 -20 mol, 1×10 -21 mol, 1×10 -22 mol, 1×10 -23 mol; 1×10 -13 mol to 1×10 -22 mol, 1×10 -11 mol to 1×10 -20 mol, 1×10 -13 mol to 1×10 -18 mol, 1×10 -15 mol to 1×10 20 or 1×10 -14 mol to 1×10 -17 mol alkaline phosphatase).

[0055] In some aspects, the luminescent intensity of the compounds of the present disclosure is described in relative light units (RLU). The term "RLU" refers to the relative light unit of a chemiluminescent signal (S) in the presence of alkaline phosphatase (ALP). In some aspects, RLU can be corrected for background chemiluminescence (B) in the absence of ALP, e.g., S minus B.

[0056] In particular, the present disclosure describes compositions comprising a combination of a dioxetane and a selected surfactant that provide unexpected emission results in an aqueous environment.The concentration of the dioxetane can be selected by a skilled artisan. Exemplary dioxetane concentrations that can be used include 0.5 mg / L to 5000 mg / L (e.g., 1 mg / L to 1000 mg / L, 1 mg / L to 500 mg / L, 25 mg / L to 2000 mg / L, 50 mg / L to 500 mg / L, 50 mg / L to 250 mg / L, 100 mg / L to 150 mg / L, 50 mg / L to 1,000 mg / L, 200 mg / L to 1200 mg / L, 50 mg / L to 500 mg / L, 50 mg / L to 150 mg / L, 100 mg / L to 200 mg / L, 100 mg / L to 500 mg / L, and 100 mg / L to 200 mg / L). In some aspects, the dioxetane concentration that can be used is at least 0.5 mg / L, at least 1 mg / L, at least 25 mg / L, at least 50 mg / L, at least 100 mg / L, at least 200 mg / L, at least 500 mg / L, at least 1000 mg / L, at least 3000 mg / L, or at least 4000 mg / L. In some aspects, the dioxetane concentration that can be used can be at most 5000 mg / L, at most 4000 mg / L, at most 3000 mg / L, at most 1000 mg / L, at most 500 mg / L, at most 200 mg / L, 100 mg / L, at most 25 mg / L, or at most 1 mg / L.

[0057] The skilled artisan can also select the concentration of the surfactant. Among the exemplary surfactants, concentrations that can be used include 5 mg / L to 25,000 mg / L (e.g., 1,000 mg / L to 10,000 mg / L, 500 mg / L to 5,000 mg / L, 50 mg / L to 10,000 mg / L, 100 mg / L to 5,000 mg / mL, 100 mg / L to 500 mg / L, 250 mg / L to 1,000 mg / L, In some aspects, the surfactant concentration that can be used can be at least 5 mg / L, at least 50 mg / L, at least 100 mg / L, at least 500 mg / L, at least 1000 mg / L, at least 3000 mg / L, at least 4000 mg / L, at least 5000 mg / L, at least 10,000 mg / L, at least 15,000 mg / L, or at least 20,000 mg / L. In some aspects, the surfactant concentration that can be used can be up to 25,000 mg / L, up to 20,000 mg / L, up to 15,000 mg / L, up to 10,000 mg / L, up to 5000 mg / L, up to 4000 mg / L, up to 3000 mg / L, up to 1000 mg / L, up to 500 mg / L, up to 100 mg / L, or up to 50 mg / L.

[0058] The concentration ratio of the dioxetane compound to the surfactant can be in the range of 5:1 to 1:10 (e.g., 1:1 to 1:5, 1:2 to 1:8; 1:1 to 1:3, 1:2 to 1:5; and 1:3 to 1:9). For example, the concentration ratio of the dioxetane compound to the surfactant can be 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0059] like Figure 1 As shown, when a fluorescein surfactant is used (e.g., as in LP530), a dioxetane compound having the following structure:

[0060]

[0061] In a relatively short period of time, it showed higher RLU than the dioxetane compound with the following structure:

[0062]

[0063] The term "LP530" as used herein refers to "Lumi-Phos 530", a product of Lumigen, Inc., which contains a dioxetane compound referred to herein as "PPD" and a fluorescein surfactant. Figure 2A and 3 As shown, the use of VPPD and aggregate The surfactant / enhancer (EXL) combination surprisingly resulted in significant differences in brightness as expressed in relative light units (RLU). The brightness of the VPPD of the surfactant / enhancer combination is over 20 times higher relative to the brightness of the VPPD combined with the fluorescein surfactant (about 9500 RLU in the EXL surfactant compared to about 380 in the fluorescein surfactant). Figure 3 .

[0064] As an added benefit, aggregation Surfactants / enhancers provide about 50-fold (and up to about 100-fold) improvement in signal-to-noise ratio. VPPD in the enhancer can detect AP10 (1.2×10 -21 mol ALP) using 10 μL of AP10 added to 100 μL of a solution of VPPD and enhancer. However, to produce essentially the same signal as LP530, 100-fold more ALP (10 μL of AP8; AP8 refers to 1.2×10 -19 mol / μL ALP). Figure 6 . Figure 7 Graph of RLU of VPPD EXL and LP530 background as a function of time, where "background" refers to the signal obtained for the VPPD composition containing EXL or LP530, each without any ALP. The signal is generated by the thermal decomposition of dioxetane when incubated at 37°C. The background signal for the composition containing VPPD and EXL is about one-third of the signal obtained for LP530. Figure 7 As shown, the lower signal without ALP (background) means that the detection limit, which is the lowest practical amount of analyte conjugated to ALP that can be reliably detected and distinguished from the background, is also lower.

[0065] In addition, if Figure 2A and 3 As shown, with the aggregation The brightness of the VPPD of the surfactant / enhancer combination is greater than that of the polymer Other dioxetanes in surfactant / enhancer combinations include, for example, PPD of surfactant / enhancer combination or with polymer VMPD of a surfactant / enhancer combination, wherein VMPD is a dioxetane compound having the following structure:

[0066]

[0067] For example, with aggregation The VMPD of the surfactant / enhancer combination has a brightness greater than LP530. In fact, as shown in these exemplary figures, The VMPD of the surfactant / enhancer combination gave a much brighter signal than LP530. VMPD in a surfactant reaches a signal maximum faster (less than 1 minute) than VPPD in the same surfactant.

[0068] These results suggest that specific dioxetane compounds The surfactant / enhancer combination provides unexpectedly improved emission results in aqueous environments.

[0069] The present disclosure provides compositions comprising a compound of formula I and its salts and at least one surfactant:

[0070]

[0071] Where R 1 and R 2 Each is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon to which they are attached, they provide C5-C 10 Cycloalkyl ring;

[0072] R 3 It is C1-C 10 Alkyl, C6-C 10 Aryl or heteroaryl;

[0073] R 4 It is C2-C 10 alkenyl;

[0074] R 5 Is H or C1-C 10 alkyl;

[0075] X is a phosphate (eg, a phosphate group of the formula -OP(O)(ONa)(ONa)).

[0076] For example, the present disclosure provides a composition comprising a compound of Formula II or a salt thereof and at least one surfactant:

[0077]

[0078] Where R 10 and R 11 are independently H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl; R 3 It is C1-C 10 Alkyl, C6-C 10 Aryl or heteroaryl; R 4 It is C2-C 10 Alkenyl; R 5 Is H or C1-C 10 In some embodiments, R 10 and R 11 is independently H or halogen.

[0079] The present disclosure provides a composition comprising a compound of formula III or a salt thereof and at least one surfactant:

[0080]

[0081] Where R 10 and R 11 are independently H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl; R 3 It is C1-C 10 Alkyl, C6-C 10 Aryl or heteroaryl; R 4 It is C2-C 10 Alkenyl; R 5 Is H or C1-C 10 In some embodiments, R 10 and R 11 is independently H or halogen.

[0082] The present disclosure also provides a composition comprising a compound of formula IV or a salt thereof and at least one surfactant:

[0083]

[0084] Where R 10 and R 11 are independently H, halogen, C1-C 10 Alkyl, C2-C 10Alkenyl, C6-C 10 Aryl; R 3 It is C1-C 10 Alkyl, C6-C 10 Aryl or heteroaryl; R 4 It is C2-C 10 Alkenyl; R 5 Is H or C1-C 10 In some embodiments, R 10 and R 11 is independently H or halogen.

[0085] The compound of formula I to IV may be a compound of the following formula or a salt thereof:

[0086]

[0087] The composition comprising formula I to IV compound can be an aqueous composition or a non-aqueous composition. The composition can be a mixture of both aqueous and non-aqueous solvents, and can include other additives (for example, ions, including magnesium ions, for example, magnesium ions derived from magnesium salts such as magnesium chloride, etc.). If the composition comprising formula I to IV compound includes magnesium ions, they can include 50mg / L to 500mg / L (for example, 50mg / L to 250mg / L, 100mg / L to 190mg / L, 150mg / L to 200mg / L or 150mg / L to 190mg / L) magnesium chloride (II) (MgCl ), but other magnesium (II) salts can also be used. In an example, magnesium ion concentration can be 0mM to 10mM, 0mM to 3mM, 0.5mM to 2mM, 0.5mM to 1mM, 1mM to 5mM or 2mM to 5mM.

[0088] In various embodiments, surfactants for use with compositions comprising compounds of Formulae I to IV include, but are not limited to Surfactants, which are also referred to herein interchangeably as Surfactants include polymers Surfactants and small molecules Surfactants. Polymerization Some examples of surfactants include Pendant polyethylene type polymers are disclosed in U.S. Pat. No. 5,393,469. For example, U.S. Pat. No. 5,393,469 describes a polymer of the general formula Surfactants:

[0089]

[0090] Wherein each A is selected from the lower alkyl, aryl or arylalkyl containing 1 to 20 carbon atoms, group F1 is a fluorescent group, m is an integer of 1 to 14, and wherein n and p are integers of about 10 to 1000. Each A group on a specific phosphorus atom can be the same group, or can be two different groups, or all three can be different. The A group group on adjacent phosphorus atoms can be the same group or can be different groups, wherein the group meets the above description. The relative position of the substituent on the aromatic ring can be a mixing of any proportion of ortho, meta, para or these three types. The fluorescent group connected can be any fluorescent agent, which can be connected to polymer chemistry, and compared with the excited state of dioxetane (for example, formula I to IV compound), its singlet electronic excited state has lower energy. Fluorescent group can enhance the chemiluminescent efficiency of dioxetane by serving as an energy acceptor, and the energy acceptor is excited and releases excitation energy in the form of light. Some examples of fluorescent agents that can be used to practice the present invention include, but are not limited to: any fluorescent dye; aromatic compounds, including polycyclic aromatic compounds, biphenyls, terphenyls, Heteroaromatic and polycyclic heteroaromatic compounds, such as acridine, coumarin, phthalocyanine, furan, Azoles, Oxadiazoles, benzothiazoles, quinolines, xanthenes, fluorescein and fluorescein derivatives, such as amidofluorescein, eosin and eosin derivatives, rhodamine and resorufin.

[0091] Exemplary Aggregation Enhancers include polyvinylbenzyltributyl chloride Polyvinylbenzyl trioctyl chloride and polyvinylbenzyltributyl chloride A copolymer having two quaternary ammonium or dicationic compounds of the group, such as those disclosed in U.S. Pat. No. 5,451,347. For example, U.S. Pat. No. 5,451,347 describes quaternary ammonium or Compounds of the group:

[0092]

[0093]

[0094] polymerization Some examples of reinforcing agents include polymers comprising repeating units (A), repeating units (B), or both:

[0095]

[0096] Wherein "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl". The molar ratio of repeating unit (A) to repeating unit (B) can be in the range of 1:100 to 100:1 (e.g., such as 100:1 to 1:1, including, for example, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 70:30, 75:25, 60:40 or 50:50). The weight average molecular weight (M w ) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M w In some embodiments, M may be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. w In some embodiments, M w In some embodiments, M w In some embodiments, M w In some embodiments, M w The copolymer may be a random copolymer, a block copolymer or a random block copolymer.

[0097] polymerization Some examples of reinforcing agents include polymers of the following formula:

[0098]

[0099] Wherein m and n are each independently an integer from 0 to 1000 (including, for example, an integer from 1 to 500, 250 to 1000, 300 to 900, 50 to 500, or 250 to 750). The ratio of m:n can be from 100:1 to 1:1, 4:1, such as 70:30, 75:25, 60:40, or 50:50; m+n is in the range of 100 to 1000 (for example, m+n can be 100 to 600, 200 to 500, or 300 to 600); the weight average molecular weight (M w) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M w In some embodiments, M may be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. w In some embodiments, M w In some embodiments, M w In some embodiments, M w In some embodiments, M w The copolymer may be a random copolymer, a block copolymer or a random block copolymer.

[0100] Exemplary Aggregation Enhancers / surfactants also include polymers containing the following structure Surfactants:

[0101]

[0102] wherein z is an integer in the range of 2 to 1000 (e.g., 2 to 500, 100 to 600, 450 to 800, 300 to 600, 2 to 250, 50 to 250, 100 to 300, 150 to 400, 175 to 400, 50 to 300, 100 to 500, 150 to 400, or 10 to 200). The weight average molecular weight (M w ) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M wIn some embodiments, M may be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. w is 70,000, 80,000, 100,000 or 220,000 g / mol. In some embodiments, M w In some embodiments, M w In some embodiments, M w In some embodiments, M w is 300,000 g / mol. Some examples of enhancers / surfactants include those comprising the following structure:

[0103] and / or

[0104] wherein z is an integer from 2 to 1000 (e.g., from 2 to 500, from 100 to 600, from 450 to 800, from 300 to 600, from 2 to 250, from 50 to 250, from 100 to 300, from 150 to 400, from 175 to 400, from 50 to 300, from 100 to 500, from 150 to 400, or from 10 to 200). The weight average molecular weight (M w ) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary aspects, M w In some embodiments, M may be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. w is 70,000, 80,000, 100,000 or 220,000 g / mol. In some embodiments, M wIn some embodiments, M w In some embodiments, M w In some embodiments, M w It is 300,000g / mol.

[0105] Exemplary Small Molecules Enhancers / surfactants include small molecule surfactants of the formula:

[0106]

[0107] in:

[0108] R 12 To R 14 Each is independently C1-C 10 alkyl;

[0109] R 15 is arylalkyl; and

[0110] X - is a counterion (e.g. chloride). 15 It may be an arylalkyl group substituted with a C2-C8 alkenyl group or a group of the formula:

[0111] where k is 0, 1, or 2; R 16 To R 18 Each is independently C1-C 16 Alkyl. 16 To R 18 Can be C4-C independently 10 Alkyl. C2-C8 alkenyl can be a group of the formula:

[0112] Where R 19 It is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl.

[0113] Small molecules Some examples of enhancers / surfactants include:

[0114]

[0115] Other surfactants contemplated herein include poly(vinylbenzyltributyl chloride )(TBE) (e.g., TBE with a molecular weight of 175,000 g / mol), polyvinylpyridine Salts (e.g., polyvinylpyridine having a molecular weight of 10,000 g / mol to 100,000 g / mol) salts), alkylammonium salts (e.g., (C8-C 20 Alkyl)2N(C1-C4 alkyl)2Cl(e.g. (C8H 17 )3N(CH3)Cl, (C 12 H 25 )2N(CH3)2Cl and (C 18 H 37 )2N(CH3)2))), alkyl-ethylene glycol ammonium salts (e.g., (C 12 -C 20 alkyl)2N(C1-C4alkyl-O)2Cl(e.g. (C 12 H 25 )2N((CH3CH2O)5)2Cl and (C 18 H 37 )2N((CH2CH2O)5)2Cl)), and aralkyl-alkylammonium salts (e.g., (C 18 H 37 )2N(CH3)(CH2Ph)Cl)).

[0116] Combinations of any of the above surfactants / enhancers (e.g., small molecules polymerization and a combination of two or more of the fluorescein surfactant / enhancer). For example, the composition may include a small molecule and Aggregation In some cases, the composition comprises a small molecule and a fluorescein surfactant / enhancer. In some cases, the composition comprises a small molecule polymerization and a fluorescein surfactant / enhancer. For example, the composition may comprise the following combination:

[0117] (a) The following polymerization

[0118] and a small molecule of the formula

[0119]

[0120] (b) The following polymerization

[0121] and a small molecule of the formula

[0122] (c) Small molecules of the formula

[0123] and a fluorescein surfactant / enhancer of the formula:

[0124]

[0125] (d) Small molecules of the formula

[0126] and

[0127] Fluorescein surfactant / enhancer of the following formula:

[0128]

[0129] (e) The following polymerization

[0130]

[0131] The following small molecule

[0132] and

[0133] Fluorescein surfactant / enhancer of the following formula:

[0134] or

[0135] (f) The following polymerization

[0136]

[0137] The following small molecule

[0138] and

[0139] Fluorescein surfactant / enhancer of the following formula:

[0140]

[0141] In various embodiments, the composition comprises a buffer solution. The buffer solution can be, but does not necessarily have to be, an alkaline buffer solution or an amine-based buffer solution. An example of an amine-based buffer is 221 buffer (a buffer based on 2-amino-2-methyl-1-propanol (AMP)), which can be obtained from Sigma-Aldrich (St. Louis, MO). Thus, the buffer solutions contemplated herein include amine-based buffer solutions containing 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (AMPD), tris(2-amino-2-(hydroxymethyl)propane-1,3-diol), TAPS (3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}propane-1-sulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine) and Tricine (N-[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]glycine) and combinations thereof, such as buffer solutions containing AMPD and AMP. Likewise, the composition may, but need not necessarily, have an alkaline pH. In some exemplary embodiments, the pH of the composition can be 4 to 12, 5 to 12, 6 to 12, 8 to 11, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5, for example, at a buffer concentration of 0.1 to 1 M, 0.1 to 0.5 M, 0.2 to 0.5 M, 0.2 to 0.8 M, 0.1 to 0.25 M, 0.2 to 0.3 M, or 0.15 M to 0.3 M. The pH of the composition can be selected based on whether the luminescence is intended to be induced immediately after the analyte induces the ALP to remove the X-group of the compound described herein, as is typically the case with alkaline pH values, or the pH of the composition can be acidic so that luminescence is induced after treatment with a base.

[0142] In various embodiments, the composition has / exhibits an RLU of greater than 2,000 RLU, greater than 4,000 RLU, greater than 6,000 RLU, greater than 8,000 RLU, greater than 9,000 RLU; at most 50,000 RLU, at most 30,000 RLU, at most 25,000 RLU, at most 20,000 RLU, at most 15,000 RLU, at most 10,000 RLU, or at most 5000 RLU; 2,000 RLU to 10,000 RLU, 4,000 RLU to 10,000 RLU, 2,000 RLU to 5,000 RLU, 2,500 RLU to 4,000 RLU, or 8,000 RLU to 10,000 RLU in the presence of ALP or ALP conjugated to an analyte of interest. RLU can be measured by any suitable means, including, for example, measuring light intensity at 37°C using a Tuner TD-20 photometer. In some aspects, 10 μL of an ALP solution (e.g., AP8) can be mixed with 100 μL of the formulated reagent. Additionally or alternatively, the composition exhibits a T 1 / 2 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 30 seconds to 4 minutes, 30 seconds to 2 minutes, 30 seconds to 1 minute, 1 minute to 3 minutes, or 1 minute to 2 minutes (e.g., at 37°C).

[0143] In various embodiments, the composition has / exhibits a peak RLU in the absence of an enzyme (e.g., ALP) of at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, or at most 10. In various embodiments, the composition has / exhibits a peak RLU in the absence of an enzyme (e.g., ALP) of at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7. In various embodiments, the composition has / exhibits a peak RLU in the absence of an enzyme (e.g., ALP) of 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 2 to 20, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 3 to 20, 3 to 15, 3 to 14, 3 to 13, or 3 to 12.

[0144] The compositions described herein comprising a dioxetane and a surfactant can include a magnesium salt in an amount of 50 mg / L to 500 mg / L (e.g., 50 mg / L to 250 mg / L, 100 mg / L to 190 mg / L, 150 mg / L to 200 mg / L, or 150 mg / L to 190 mg / L) magnesium (II) chloride (MgCl), although other magnesium (II) salts can also be used.

[0145] The present disclosure also provides a method for detecting an analyte (e.g., ALP or ALP conjugated to an analyte of interest) in a sample, comprising contacting the sample with one or more of the compounds or salts thereof described herein or a composition comprising one or more of the compounds or salts thereof described herein, and then monitoring the luminescence of the sample. In various embodiments, the method comprises measuring the intensity of the resulting luminescence and correlating the intensity with at least one of the presence and concentration of the analyte (e.g., ALP or ALP conjugated to an analyte of interest). In various embodiments, the concentration of ALP detected by the method is at most 1×10 -18 , 1×10 -19 , 1×10 -20 or 1×10 -21 In various embodiments, the ALP concentration detected by the method is at least 1×10 -22 mol / μL or at least 1×10 -23 In various embodiments, the ALP concentration detected by the method is 1×10 -23 mol / μL to 1×10 -18 mol / μL (e.g., 1×10 -23 mol / μL to 1×10 - 19 mol / μL; 1×10 -22 mol / μL to 1×10 -18 mol / μL; or 1×10 -23 mol / μL to 1×10 -20 In various embodiments, the method detects at least 1×10 -22 mol ALP or at least 1×10 -21 In various embodiments, the method detects up to 1×10 -17 , up to 1×10 -18 , up to 1×10 -19 or up to 1×10 -20 In various embodiments, the method detects ALP in the range of 1×10 -22 mol ALP to 1×10 -17 mol ALP (e.g., 1×10 -22 mol ALP to 1×10 -18 mol ALP; 1×10 -22 mol ALP to 1×10 -19 mol ALP; or 1×10 -21 mol ALP to 1×10 -17 mol ALP).

[0146] In some embodiments, the method further comprises increasing the pH of the sample. For example, the pH can be adjusted to 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher.

[0147] In various embodiments, the analyte is detected in 10 minutes or less, 6 minutes or less, 4 minutes or less, 2 minutes or less, 1 minute or less, 55 seconds or less, 45 seconds or less, or 30 seconds or less.

[0148] The present disclosure also provides a kit for determining at least one of the presence and concentration of an analyte. In some embodiments, the kit comprises any one or more of the compounds described herein, an olefin precursor thereof, a salt thereof, or a composition comprising them. The kit may comprise instructions according to the methods described herein.

[0149] The compounds and compositions described herein can be triggered directly by adding an analyte, such that a signal identifying at least one of the presence and concentration of the probe and analyte is generated, or can be triggered in a two-step process, one step involving contacting the analyte and the other step involving raising the pH.

[0150] The compounds of the present disclosure can be configured as probes to detect ALP or ALP conjugated to an analyte of interest.

[0151] It is desirable that the compound emits light in as short a period of time as possible after being triggered by the analyte to provide as strong a signal as possible.When chemiluminescence is emitted gradually over a period of time, the light intensity (photons / second) decreases and detection sensitivity can suffer.

[0152] The rate of increase in luminescence or rise time can be determined based on the time to reach maximum emission (t max ) or emission half-life (T 1 / 2 ) to describe.

[0153] The compounds described herein can be used as enzyme substrates for alkaline phosphatase (ALP) and the like. Without being limited by theory, one exemplary mechanism involves the ALP enzyme hydrolyzing X, where X is a phosphate group, to provide a phenol that is immediately deprotonated due to the alkaline environment of the solution (e.g., pH 9.7 buffer). The formation of the oxyanion triggers the decomposition of 1,2-dioxetane into two compounds: 2-adamantanone and a phenyl ester in an excited state. The excited phenyl ester then immediately decays to the ground state by releasing light.

[0154] The resulting light intensity is a linear function of the amount of enzyme. Therefore, the compounds described herein can be used to detect marker enzymes used in assays. For example, the steps of a chemical process in which a dioxetane provides light can be described according to the following steps: (i) X+S→X+S′ (ii) S′→P ＊ and (iii) P ＊ →P+light. Step (i) represents the catalytic conversion of the substrate, where X is an enzyme or other component that converts the substrate (S) into its activated form (S'), step (ii) represents the degradation of the activated substrate into a transient excited species (P*), and step (iii) represents the decay of the excited species to the ground state (P) and emission of light. The light intensity is the product of the catalytic conversion of the substrate in step (i) and the lifetime of the resulting light-generating compound P* in step (ii). Step (ii) is typically first order, with a rate constant k, and can be characterized by its half-life: T 1 / 2 =(ln 2) / k. Step (iii) is very short compared to the other steps and usually has no significant effect on the reaction kinetics.

[0155] The chemiluminescence intensity / time curve includes an initial rise period of emission intensity and a subsequent steady-state intensity period. ＊ The slow first-order reaction of corresponds to a prolonged rise time, since it takes longer to reach the steady-state concentration of S'. ＊ The rapid response corresponds to a shorter initial rise period and thus provides a rapid rise. For enzymatic chemiluminescent reactions, the intensity is usually kept at a high level. Lack of stable intensity indicates substrate exhaustion or subsequent enzyme inactivation. Enzyme-generated chemiluminescent detection provides flexibility in the measurement process, because the light intensity at any time point can be related to the amount of enzyme, but the enzyme-generated process may have disadvantages, for example, due to the size and "sticky" nature of the enzyme label. However, for maximum sensitivity, during the steady-state intensity period at maximum intensity (I max ) is best measured at or near the

[0156] Compounds of the present disclosure can generally be prepared, for example, according to the synthetic methods described in Scheme 1.

[0157]

[0158] Solution 1

[0159] wherein G is any suitable oxygen protecting group.

[0160] Briefly, 2-adamantanone and 3-substituted benzoate can be coupled together by subjecting them to McMurry reaction conditions involving an oxophilic titanium and a reducing agent. The resulting olefin can be further modified, for example by removing or replacing the protecting group G or further modifying R. 4Next, the olefin is subjected to photooxidation reaction conditions to provide a 1,2-dioxetane product. In various embodiments, R 4 and X are as described in any of the various embodiments of the present application. In some embodiments, R 10 and R 11 is H. In some embodiments, R 3 is a substituted or unsubstituted alkyl group.

[0161] The term "intensity" (e.g., light intensity or luminous intensity) as used herein refers to the emission rate in photons / second. Intensity can be measured using a photometer. A photometer is a photodetector in a housing that excludes ambient light. Any suitable photometer can be used, including photomultiplier tubes and / or photodiodes.

[0162] The term "luminescence rate" refers to the rate at which luminescence increases, ie, the change in light intensity over time.

[0163] As used herein, the term "sensitivity" refers to the lowest level of signal at which the analyte or product being measured can be reproducibly detected.

[0164] As used herein, the term "alkyl" refers to a substituted or unsubstituted straight chain, branched or cyclic, saturated monovalent or divalent group having 1 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 1 to 6 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 1 to 3 carbon atoms. 20 Some examples of alkyl groups include those having 1 to 8 carbon atoms, such as methyl (i.e., CH3), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. 20 Some examples of alkyl groups include isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, and isopentyl. 20Some examples of alkyl groups include those having 1 to 6 carbon atoms, such as -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-. Some examples of branched divalent alkyl groups include -CH(CH3)CH2- and -CH2CH(CH3)CH2-. Some examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, and adamantyl. Cycloalkyl groups also include substituted and unsubstituted polycyclic cycloalkyl groups, such as, but not limited to, norbornyl, adamantyl, bornyl, bornenyl, isobornenyl, and carenyl, as well as fused rings, such as, but not limited to, decahydronaphthyl, etc. For example, cycloalkyl includes adamantyl substituted with one, two, three, four or more substituents, such as at the tertiary bridgehead position of a methylene bridge. In some embodiments, alkyl includes a combination of substituted and unsubstituted alkyl. As an example, alkyl and (C1) alkyl include methyl and substituted methyl. As a specific example, (C1) alkyl includes benzyl. As another example, alkyl may include methyl and substituted (C2-C8) alkyl. Alkyl may also include substituted methyl and unsubstituted (C2-C8) alkyl. In some embodiments, alkyl may be methyl and C2-C8 straight chain alkyl. In some embodiments, alkyl may be methyl and C2-C8 branched chain alkyl. The term methyl is understood to be -CH3, which is unsubstituted. The term methylene is understood to be -CH2-, which is unsubstituted. For comparison, the term (C1) alkyl is understood to be substituted or unsubstituted -CH3 or substituted or unsubstituted -CH2-. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, such as cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxyl, cyano, carboxyl, nitro, sulfenyl, alkoxy, and halogen. As another example, representative substituted alkyl groups can be substituted with one or more fluorine, chlorine, bromine, iodine, amino, amide, alkyl, alkoxy, alkylamide, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxyl, haloalkyl, hydroxyl, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, sulfenyl, alkylsulfenyl, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylaminoacyl. In some embodiments, representative substituted alkyl groups can be substituted from a group of groups including amino, hydroxyl, cyano, carboxyl, nitro, thio and alkoxy groups, but excluding halogen groups. Therefore, in some embodiments, alkyl groups can be substituted with non-halogen groups.For example, representative substituted alkyl groups can be substituted with fluoro groups, substituted with bromo groups, substituted with halogens other than bromo groups, or substituted with halogens other than fluorine. In some embodiments, representative substituted alkyl groups can be substituted with one, two, three or more fluoro groups, or they can be substituted with one, two, three or more non-fluoro groups. For example, alkyl groups can be trifluoromethyl, difluoromethyl or fluoromethyl groups, or alkyl groups can be substituted alkyl groups other than trifluoromethyl, difluoromethyl or fluoromethyl groups. Alkyl groups can be haloalkyl groups, or alkyl groups can be substituted alkyl groups other than haloalkyl groups.

[0165] As used herein, the term "alkenyl" refers to a substituted or unsubstituted straight, branched or cyclic saturated monovalent or divalent group having at least one carbon-carbon double bond and 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The double bond may be trans or cis oriented. The double bond may be terminal or internal. The alkenyl may be connected by an alkenyl moiety containing a double bond (e.g., vinyl, propene-1-yl, and butene-1-yl), or the alkenyl may be connected by an alkenyl moiety not containing a double bond (e.g., pentene-4-yl). In the case specified, the parent moiety is understood to be connected to the alkenyl at the ethylene position of the double bond rather than at the non-ethylene position. For example, when an aromatic ring is substituted with a π-conjugated olefinic group, it is understood to be substituted at an ethylene position rather than a non-ethylene position. As another example, an aromatic ring substituted with a π-conjugated propenyl group would be understood to be propen-1-yl or propen-2-yl rather than propen-3-yl. 20 Some examples of alkenyl groups include those having 1 to 8 carbon atoms, such as ethenyl, propenyl, propen-1-yl, propen-2-yl, butenyl, buten-1-yl, buten-2-yl, sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, heptenyl, and octenyl. 20 Some examples of alkenyl groups include isopropenyl, isobutenyl, sec-butenyl, tert-butenyl, neopentenyl, and isopentenyl. 20Some examples of )alkenyl groups include those having 2 to 6 carbon atoms, such as -CHCH-, -CHCHCH2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-. Some examples of branched divalent alkyl groups include -C(CH3)CH- and -CHC(CH3)CH2-. Some examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl, and cyclooctenyl. For example, the alkenyl group may be vinyl and substituted vinyl. For example, the alkenyl group may be vinyl and substituted (C3-C8)alkenyl. The alkenyl group may also include substituted vinyl and unsubstituted (C3-C8)alkenyl. Representative substituted alkenyl groups may be substituted one or more times by any of the groups listed herein, such as monoalkylamino, dialkylamino, cyano, acetyl, amide, carboxyl, nitro, alkylthio, alkoxy, and halogen groups. As another example, representative substituted alkenyl can be substituted by one or more fluorine, chlorine, bromine, iodine, amino, amide, alkyl, alkoxy, alkylamide, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxyl, haloalkyl, hydroxyl, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, sulfenyl, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamide. In some embodiments, representative substituted alkenyl can be substituted from a group of groups, including monoalkylamino, dialkylamino, cyano, acetyl, amide, carboxyl, nitro, alkylthio and alkoxy, but not including halogen groups. Therefore, in some embodiments, alkenyl can be substituted by non-halogen groups. In some embodiments, representative substituted alkenyl can be substituted by fluoro group, substituted by bromo group, substituted by halogen other than bromo group, or substituted by halogen other than fluorine. For example, alkenyl can be 1-fluorovinyl, 2-fluorovinyl, 1,2-difluorovinyl, 1,2,2-trifluorovinyl, 2,2-difluorovinyl, trifluoropropylene-2-yl, 3,3,3-trifluoropropylene, 1-fluoropropylene, 1-chlorovinyl, 2-chlorovinyl, 1,2-dichlorovinyl, 1,2,2-trichlorovinyl or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenyl can be substituted by one, two, three or more fluoro groups, or they can be substituted by one, two, three or more non-fluoro groups.

[0166] As used herein, the term "alkynyl" refers to substituted or unsubstituted straight and branched chain alkyl groups, except that at least one triple bond is present between two carbon atoms. Thus, alkynyl has 2 to 50 carbon atoms, 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples include, but are not limited to, ethynyl, propynyl, propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, hexynyl, examples include, but are not limited to

[0167] -C≡CH,-C≡C(CH3),-

[0168] C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3), etc.

[0169] The term "aryl" as used herein refers to a substituted or unsubstituted monovalent group derived by removing a hydrogen atom from an aromatic hydrocarbon, which is a cyclic aromatic hydrocarbon having 6 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 6 to 10 carbon atoms, or 6 to 8 carbon atoms. 20 ) Some examples of aryl groups include phenyl, naphthyl, azulenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylene, pyrenyl, naphthphenyl, triphenylene, anthracenyl. Examples include substituted phenyl, substituted naphthyl, substituted azulenyl, substituted biphenyl, substituted indanyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenylene, substituted pyrenyl, substituted naphthphenyl, substituted triphenylene, and substituted anthracenyl. Examples also include unsubstituted phenyl, unsubstituted naphthyl, unsubstituted azulenyl, unsubstituted biphenyl, unsubstituted indanyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl, unsubstituted triphenylene, unsubstituted pyrenyl, unsubstituted naphthphenyl, unsubstituted triphenylene, and unsubstituted anthracenyl. Aryl groups include phenyl as well as non-phenyl aryl groups. It is clear from these examples that the term (C6-C 20 )Aryl includes monocyclic and polycyclic (C6-C 20 ) aryl, including fused and non-fused polycyclic (C6-C 20 )aryl.

[0170] The term "heterocyclic group" as used herein refers to a substituted aromatic, unsubstituted aromatic, substituted non-aromatic and unsubstituted non-aromatic ring containing 3 or more atoms in the ring, one or more of which are heteroatoms, such as but not limited to N, O and S. The term "heteroaryl" is a fully aromatic heterocyclic group, and is therefore a subset of the term heterocyclic group. The term "heterocyclic alkenyl" refers to a heterocyclic group containing an alkene in a non-aromatic ring, so that the alkene is the point of connection with the parent part. Therefore, the heterocyclic group can be a heterocyclic alkyl, a heterocyclic alkenyl or a heteroaryl, or if it is polycyclic, it can be any combination thereof. In some embodiments, the heterocyclic group comprises 3 to 20 ring members, while other such groups have 3 to 15 ring members. In some embodiments, the heterocyclic group includes a heterocyclic group comprising 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6) or 6 to 8 carbon atoms (C6-C8). The heterocyclic radical referred to as C2 heterocyclic radical can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms, etc. Similarly, a C4 heterocyclic radical can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, etc. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. The heterocyclic radical ring can also contain one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclic radical. The phrase heterocyclic radical includes fused ring materials, including those containing fused aromatic and non-aromatic groups. Representative heterocyclic radicals include, but are not limited to, piperidinyl, pyrrolidinyl, piperazinyl, and morpholinyl. For example, heterocyclic radicals include, but are not limited to:

[0171]

[0172] Where X 1 Represents H, (C1-C 20 ) alkyl, (C6-C 20 ) aryl or amine protecting group (such as tert-butyloxycarbonyl group), and the heterocyclyl group can be substituted or unsubstituted. Representative heteroaryl groups include furanyl, pyridyl, pyrazinyl, pyrimidinyl, triazine, thienyl, tetrahydrofuranyl, pyrrolyl, Azolyl, imidazolyl, triazolyl, tetrazolyl, benzo In some embodiments, the heteroaryl group is a 5-membered heteroaryl group. In some embodiments, the heteroaryl group is not pyridine, pyrimidine, pyridazine, pyrazine, or a fused derivative thereof. A π-excess heteroaryl group is an electron-rich heteroaryl group such that it can be used as an electron-donating group. Some examples of π-excess heteroaryl groups are furan, thiophene, indole, pyrrole, benzofuran, and benzothiophene.

[0173] The term "alkoxy" used herein refers to an oxygen atom connected to an alkyl group (including cycloalkyl) defined herein. Some examples of straight chain alkoxy include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, etc. Some examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, etc. Some examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc. Alkoxy may include 1 to 12 to 20 or 12 to 40 carbon atoms bonded to an oxygen atom, and may also include double bonds or triple bonds, and may also include heteroatoms. Therefore, alkoxy also includes an oxygen atom connected to an alkynyl (alkyenyl) group and an oxygen atom connected to an alkynyl group. For example, allyloxy is an alkoxy group within the meaning of this article. Methoxyethoxy is also an alkoxy group within the meaning of this article, as is the case with methylenedioxy in the case where two adjacent atoms of the structure are replaced by it.

[0174] The term "aryloxy" as used herein refers to an oxygen atom attached to an aryl group as defined herein. The point of substitution on the parent moiety is at the oxygen atom.

[0175] The term "arylcarbonyl" as used herein refers to a carbonyl (CO) group appended to an aryl group as defined herein. The point of substitution on the parent moiety is at the carbonyl.

[0176] The term "heteroarylcarbonyl" as used herein refers to a carbonyl (CO) group appended to a heteroaryl group as defined herein. The point of substitution on the parent moiety is at the carbonyl.

[0177] The term "arylalkyl" as used herein refers to an alkyl group as defined herein, wherein a hydrogen or carbon bond of the alkyl group is replaced with a bond to an aryl group as defined herein. Representative arylalkyl groups include benzyl, biphenylmethyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indanyl. Arylalkenyl is an alkenyl group as defined herein, wherein a hydrogen or carbon bond of the alkenyl group is replaced with a bond to an aryl group as defined herein. The point of substitution of the parent moiety is at the alkyl group.

[0178]

[0046] As used herein, the terms "halo," "halogen," or "halide" group, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0179] The term "amino" as used herein refers to -NH2, -NHR, -NR2, -NR3 + Substituents of the form wherein each R is independently selected and the protonated form of each (except -NR3 +), which cannot be protonated. Therefore, any compound substituted with an amino group can be considered an amine. "Amino" within the meaning of this article can be a primary amino group, a secondary amino group, a tertiary amino group or a quaternary amino group. "Alkylamino" groups include monoalkylamino, dialkylamino and trialkylamino.

[0180] The term "acyl" as used herein refers to a group comprising a carbonyl moiety, wherein the group is bonded through a carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which may be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, and the like.

[0181] As used herein, the term "formyl" refers to a group containing an aldehyde moiety. The point of substitution on the parent moiety is at the carbonyl.

[0182] The term "alkoxycarbonyl" used herein refers to a group containing a carbonyl moiety, wherein the group is bonded by a carbonyl carbon atom. The carbonyl carbon atom is also bonded to an oxygen atom, which is further bonded to an alkyl group. Alkoxycarbonyl also includes groups in which the carbonyl carbon atom is also bonded to an oxygen atom, which is further bonded to an alkynyl group. Alkoxycarbonyl also includes groups in which the carbonyl carbon atom is also bonded to an oxygen atom, which is further bonded to an alkynyl group. In another case, it is included in the definition of the alkoxycarbonyl of the term as defined herein, and is also included in the term "aryloxycarbonyl", in which the carbonyl carbon atom is bonded to an oxygen atom, which is bonded to an aryl group rather than to an alkyl group.

[0183] The term "alkylamide" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded through a carbonyl carbon atom. The carbonyl carbon atom is also bonded to a nitrogen group, which is bonded to one or more alkyl groups. In another case of an alkylamide, also as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom, which is bonded to one or more aryl groups instead of one or more alkyl groups, or is bonded to one or more aryl groups in addition to being bonded to one or more alkyl groups. In another case of an alkylamide, also as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom, which is bonded to one or more alkenyl groups instead of one or more alkyl and / or aryl groups, or is bonded to one or more alkenyl groups in addition to being bonded to one or more alkyl and / or aryl groups. In the case of another alkylamido term, also as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more alkynyl groups instead of, or in addition to, being bonded to one or more alkyl, alkenyl and / or aryl groups.

[0184] The term "carboxyl" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded through a carbonyl carbon atom. The carbonyl carbon atom is also bonded to a hydroxyl or an oxyanion to produce a carboxylic acid or a carboxylate. The carboxyl group also includes both the protonated form and the salt form of the carboxylic acid. For example, the carboxyl group can be understood as COOH or CO2H.

[0185] The term "alkylthio" as used herein refers to a sulfur atom attached to an alkyl, alkenyl or alkynyl group as defined herein. The point of substitution on the parent moiety is at the sulfur atom.

[0186] The term "arylthio" as used herein refers to a sulfur atom attached to an aryl group as defined herein. The point of substitution on the parent moiety is at the sulfur atom.

[0187] The term "alkylsulfonyl" as used herein refers to a sulfonyl group appended to an alkyl, alkenyl or alkynyl group as defined herein. The point of substitution on the parent moiety is at the sulfonyl group.

[0188] The term "alkylsulfinyl" as used herein refers to a sulfinyl group appended to an alkyl, alkenyl or alkynyl group as defined herein. The point of substitution on the parent moiety is at the sulfinyl group.

[0189] The term "dialkylaminosulfonyl" as used herein refers to a sulfonyl group attached to a nitrogen that is further attached to two alkyl groups as defined herein, and which may optionally be linked together to form a ring with the nitrogen. The term also includes groups where the nitrogen is further attached to one or two alkenyl groups rather than alkyl groups. The point of substitution of the parent moiety is at the sulfonyl group.

[0190] The term "dialkylamino" as used herein refers to an amino group connected to two alkyl groups as defined herein, and which may optionally be linked together to form a ring with the nitrogen. The term also includes groups wherein the nitrogen is further connected to one or two alkenyl groups rather than alkyl groups. The point of substitution of the parent moiety is at the nitrogen atom.

[0191] The term "dialkylamido" as used herein refers to an amide group connected to two alkyl groups as defined herein, and which may optionally be linked together to form a ring with nitrogen. The term also includes groups in which the nitrogen is further connected to one or two alkenyl groups rather than alkyl groups. The point of substitution of the parent moiety is at the amide group.

[0192] Each of the multiple substituents described herein may be substituted or unsubstituted. As used herein, the term "substituted" (e.g., in the context of "optionally substituted arylalkyl") refers to a group substituted with one or more groups (substituents) including, but not limited to, deuterium (D), halogen (e.g., F, Cl, Br, and I), R, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, methylenedioxy, ethylenedioxy, (C3-C 20 )Heteroaryl, N(R)2, Si(R)3, SR, SOR, SO2R, SO2N(R)2, SO3R, P(O)(OR)2, OP(O)(OR)2, C(O)R, C(O)C(O)R , C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, C(O)N(R)OH, OC(O)N(R)2, C(S)N(R)2, (CH2) 0-2 N(R)C(O)R, (CH2) 0-2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N (R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R or C(=NOR)R, where R can be hydrogen, (C1-C 20 )alkyl or (C6-C 20) aryl. Substituted also includes groups substituted by one or more groups, and the one or more groups include but are not limited to the following groups: fluorine, chlorine, bromine, iodine, amino, amide, alkyl, alkoxy, alkylamide, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxyl, haloalkyl, hydroxyl, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, sulfenyl, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamide. When there are two or more adjacent substituents, the substituents can be connected to form a carbocyclic ring or a heterocyclic ring. Such adjacent groups can have an ortho position or a germinal relationship, or they can be adjacent on a ring arranged in an ortho position, for example. Each substituted example is understood to be independent. For example, a substituted aryl can be substituted by bromine, and a substituted heterocyclic ring on the same compound can be substituted by an alkyl. It is contemplated that the substituted group may be substituted with one or more non-fluorinated groups. As another example, the substituted group may be substituted with one or more non-cyano groups. As another example, the substituted group may be substituted with one or more groups other than haloalkyl. As yet another example, the substituted group may be substituted with one or more groups other than tert-butyl. As yet another example, the substituted group may be substituted with one or more groups other than trifluoromethyl. As other examples, the substituted group may be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromine, other than chlorine, other than amide, other than halogen, other than benzodioxepinyl, other than polycyclic heterocyclic groups, other than polycyclic substituted aryls, other than methoxycarbonyl, other than alkoxycarbonyl, other than thienyl, or other than nitrophenyl, or with groups satisfying such a combination of descriptions. In addition, substituted should also be understood to include fluorine, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromine, chlorine, amide, halogen, benzodioxepinyl, polycyclic heterocyclic radicals, polycyclic substituted aryls, methoxycarbonyl, alkoxycarbonyl, thienyl, and nitrophenyl. In multiple embodiments, the substituted group can be substituted with a group other than a carbonyl-containing group, nitro, cyano, sulfinyl, sulfonyl, or a halogen-containing group. In multiple embodiments, the substituted group can be substituted with a group other than an electron-withdrawing group. Some substituted groups in certain embodiments can be substituted only with one or more electron-donating groups.

[0193] The term alcohol protecting group used herein refers to a substituent on an oxy group that renders oxygen inert to a variety of conditions in which alcohols are typically reacted, but is easily removed when subjected to certain conditions. Alcohol protecting groups as described herein will typically improve the stability of the dioxetane moiety, and will promote the decomposition of the dioxetane after they are removed. Therefore, alcohol protecting groups include phosphates such as PO3Na2, PO3Cl2 and PO3H2, glycosyl groups, dinitrobenzenesulfonylaminobenzyl groups and other groups that can be enzymatically hydrolyzed to provide unprotected alcohols. Some alcohol protecting groups are described in Theodora W.Greene, Peter G.M.Wuts (1999). Protecting Groups in Organic Synthesis (3ed.). J.Wiley. Alcohol protecting groups include acetyl, benzoyl, benzyl, methoxyethoxymethyl, dimethyltrityl, methoxymethyl, methylthiomethyl, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl, trialkylsilyl, trialkylsilyloxymethyl, dialkylarylsilyl, glycosyl, pyranyl, galactosyl and ethoxyethyl. Alcohol protecting groups also include groups in which alcohol is further substituted with a fragmentable linker substituted with a protecting group, wherein when such a protecting group is deprotected, the linker is fragmented and eliminated from the alcohol. The following compounds are other examples of alcohols substituted with alcohol protecting groups:

[0194]

[0195] In various embodiments, the protecting group G can be an enzyme cleavable group, wherein the cleavable group is removed by the analyte of interest, for example, in the presence of an enzyme capable of cleaving the enzyme cleavable group, to provide an unstable phenolate-dioxetane species that subsequently decomposes and emits light. For example, G can be a peptide moiety consisting of two or more amino acid residues that can be cleaved by a specific enzyme.

[0196] In some cases, the compounds described herein (eg, compounds of Formulas I to IV) may contain chiral centers. All diastereomers and racemates of the compounds described herein are contemplated herein.

[0197] The term "salt" as used herein refers to a derivative of a disclosed compound, wherein the parent compound is modified by preparing an acid or base salt thereof. Some examples of salts include alkali salts and alkaline earth salts of ionized forms of the disclosed compounds. For example, lithium salts, sodium salts, potassium salts, calcium salts, or magnesium salts. The disclosed compound may be a salt comprising a cationic metal and an anionic organic compound, such as a compound having an oxyanion and a sodium cation. Some examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups (e.g., amines); and alkaline or organic salts of acidic groups (e.g., carboxylic acids). Pharmaceutically acceptable salts include conventional nontoxic salts or quaternary ammonium salts of the parent compound formed, for example, by nontoxic inorganic or organic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isethionic acid, and the like.

[0198] Salts can be synthesized by conventional chemical methods from parent compounds containing basic or acidic moieties. In some cases, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric (or greater) amount of a suitable base or acid in water or in an organic solvent or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts is found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985.

[0199] Unless otherwise indicated, all numbers used in this specification and claims to represent the amount of components, molecular weight, etc. should be understood to be modified by the term "about" in all cases. The term "about" used herein in connection with a measured amount refers to a variation of the measured amount, as would be expected by a person skilled in the art when making measurements and taking a level of care commensurate with the purpose of the measurement and the accuracy of the measuring equipment used. Therefore, unless otherwise indicated to the contrary, the numerical parameters set forth in this specification and claims are approximate values, which may vary according to the desired properties attempted to be obtained by the present invention. At least, and without attempting to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted according to the reported numerical value of the significant figures and by applying general rounding techniques.

[0200] Values ​​expressed in the form of ranges should be interpreted in a flexible manner to include not only the values ​​explicitly stated as range limits, but also all individual values ​​or sub-ranges encompassed within the range, as if each value and sub-range were explicitly stated. For example, a range of "0.1% to 5%" or "0.1% to 5%" should be interpreted as including not only 0.1% to 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and sub-ranges within the indicated range (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). In addition, the term "range of..." or "within the range of..." (and similar expressions) includes the endpoints of the range. Here, for example, "up to" a number (e.g., "up to 50") includes that number (e.g., 50).

[0201] In this document, nouns without quantifiers are used to include one or more than one, unless the context clearly dictates otherwise. Unless otherwise indicated, the term "or" is used to refer to a non-exclusive "or". In addition, the term "and / or" means one or all of the listed elements, or a combination of any two or more of the listed elements.

[0202] In addition, it should be understood that the words or terms used herein and not otherwise defined are for descriptive purposes only and are not restrictive. Any use of section headings is intended to assist in reading the document and should not be construed as limiting. In addition, information related to section headings may appear within or outside that particular section. In addition, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference as a whole, as if separately incorporated by reference. If there is an inconsistency between the use of this document and those documents incorporated by reference, the usage in the incorporated references should be considered as a supplement to the usage of this document; for conflicting inconsistencies, the usage in this document should prevail.

[0203] In the methods described herein, except when the time or order of operations is clearly recorded, the steps can be performed in any order without departing from the principles of the present invention. In addition, unless the clear claim language records that a specific step is performed separately, the specific steps can be performed simultaneously. For example, the step of performing X claimed and the step of performing Y claimed can be performed simultaneously in a single operation, and the resulting process will fall within the text scope of the claimed method.

[0204] The term "comprises" and its variations do not have a limiting meaning where these terms appear in this specification and claims. Such terms will be understood to imply the inclusion of the stated steps or elements, or groups of steps or elements, without excluding any other steps or elements, or groups of steps or elements.

[0205] "Consisting of is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of is meant to include any elements listed after the phrase, and be limited to other elements that do not interfere with or contribute to the specified activity or action of the listed elements in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

[0206] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0207] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Therefore, the description is not intended to and should not be interpreted as being limited to the examples given, but should be granted the full scope of protection provided by the appended claims and their equivalents. In addition, some features of the present disclosure may be used without corresponding use of other features. Therefore, the purpose of providing the aforementioned description or illustrative embodiments is to illustrate the principles of the present disclosure rather than to limit it, and may include modifications and arrangements thereto.

[0208] Example

[0209] The invention may be better understood by reference to the following examples which are provided by way of illustration.The invention is not limited to the examples given herein.

[0210] General Approach

[0211] Various compounds of the present disclosure can be synthesized according to various methods, including but not limited to the synthesis methods described in PCT International Application WO1996 / 015122A1, US Patent No. 4,962,192, or US Patent No. 5,004,565.

[0212] Chemiluminescence (emission) intensity can be measured using a Turner Designs (Sunnyvale, CA) Model TD-20e luminometer, a BMG Labtech luminescence plate reader, or a charge-coupled device (CCD) camera luminometer, or any other suitable light intensity measuring device. In the examples listed below, solutions containing different concentrations of alkaline phosphatase (e.g., AP4, whose ALP concentration was 1.2×10 -15mol / μL; AP6, whose ALP concentration is 1.2×10 -17 mol / μL; AP8, its ALP concentration is 1.2×10 -19 mol / μL; and AP9, whose ALP concentration was 1.2×10 -20 mol / μL). Compounds and surfactants were tested at their approximate optimal concentrations. Figure 5 As shown, as a function of ALP concentration, the aggregation The RLU of VPPD in surfactant was essentially linear (100 μL of initial solution was combined with 10 μL of ALP solution, data collected over 240 seconds).

[0213] Nuclear magnetic resonance (NMR) spectra were obtained in solutions of D2O and CDCl3 using a 400 MHz spectrometer.

[0214] Amine-based buffer "221" or "Sigma-221" can be obtained from Sigma-Aldrich (St. Louis, MO). However, other commercially available buffers can be used in the compositions described herein.

[0215] Example 1

[0216] Dioxetane compounds [ PPD] was obtained from Lumigen, Inc. and has the following structure:

[0217]

[0218] The synthesis of PPD is described in WO2021 / 086977 (Example 12).

[0219] 1 H NMR (400MHz, D2O) δppm7.40-7.15(m,4H),3.24(s,3H),2.89(s,1H),2.28(s,1H),1.90-1.50(m,10H),1.28(d,J=13.2Hz,1H),0.99(d,J=10Hz,1H).

[0220] Example 2

[0221] VPPD, which has the following structure:

[0222]

[0223] was obtained using the method described in WO2021 / 086977 (Example 16).

[0224] 1 H NMR(400MHz,D2O)δppm7.67(d,J=8.4Hz,1H),7.66(br,1H),7.26(br,1H),7.16-6.06(m,1H),5.88(d,J=16Hz,1H),5.37(d ,J=12.4Hz,1H),3.27(s,3H),2.88(s,1H),2.30(s,1H),1.84-1.56(m,10H),1.28(d,J=9.2Hz,1H),1.05(d,J=12.8Hz,1H).

[0225] Example 3

[0226] VMPD, which has the following structure:

[0227]

[0228] was obtained using the method described in WO2021 / 086977 (Example 18).

[0229] 1 H NMR (400MHz, D2O) δppm (D2Oppm): 7.51-7.41 (m, 2H), 7.29-7.20 (m, 1H), 5.66 (d, J = 17.2Hz, 1H), 5.19 (d, J = 11.2Hz, 1H),3.07(s,3H),2.72(s,1H),2.36(s,3H),1.99(s,1H),1.95-1.38(m,10H),1.19-1.15(m,1H),0.92-0.88(m,1H).

[0230] Example 4

[0231] Initial solutions of the dioxetane compounds of Examples 1 to 3 and surfactants were prepared in a suitable buffer (e.g., 221 buffer). For each example, the dioxetane concentration used was a concentration in the range of 50 mg / L to 500 mg / L. Figure 2B Different concentrations of dioxetane VPPD (in the range of 50 mg / L to 500 mg / L) are shown while keeping the surfactant EXL concentration constant. The surfactant concentration used was in the range of 100 mg / L to 500 mg / L. Figure 2CDifferent concentrations of surfactant EXL are shown (in the range of 100 mg / L to 500 mg / L), while keeping the dioxetane VPPD concentration constant. The concentration ratio of the dioxetane compound to the surfactant is a single ratio in the range of 5:1 to 1:10. In one example, the dioxetane concentration (e.g., VPPD) is a concentration of 100 mg / L to 150 mg / L; the surfactant concentration (e.g., EXL) is a concentration of 200 mg / L to 300 mg / L; the dioxetane (e.g., VPPD) to the surfactant (e.g., EXL) is a ratio in the range of 1:1 to 1:3. The tested composition contains 50 mg / L to 500 mg / L of magnesium (II) chloride (MgCl2), but other magnesium (II) salts may also be used.

[0232] Next, 100 μL of the initial solution was combined with 10 μL of alkaline phosphatase (AP8) solution at 37° C. After combining the compounds with the alkaline phosphatase solution, the intensity of the chemiluminescence was measured over time. Figure 1 and 2A A graph showing the light emission intensity (Relative Luminescence Units (RLU) over time) of, in particular, 100 μL of the initial solution in combination with 10 μL of the AP8 solution is provided, wherein Figure 1 RLU data for LP530 is shown as a function of time. The fluorescein surfactant in LP530 combined with cetyltrimethylammonium bromide (CTAB) has the following formula:

[0233] wherein p is an integer from 2 to 10, such as an integer from 5 to 9, or from 5 to 7.

[0234] In a separate experiment, EXL surfactant was used. Figure 2A A graph showing light emission intensity (Relative Luminescence Units (RLU)) over time is provided. EXL surfactants comprise repeating units (A), repeating units (B), or both:

[0235]

[0236] Wherein "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl". The molar ratio of the repeating unit (A) to the repeating unit (B) used is 4:1. Figure 2B and 2C is a graph of RLU over time at various VPPD concentrations (50 mg / L to 500 mg / L) and various EXL surfactant concentrations (100 mg / L to 500 mg / L). In both experiments, solutions of VPPD and surfactant were mixed with 6 μL AP8 and then incubated at 37°C. Figure 2B and 2CIt is shown that different concentrations of dioxetane VPPD (while keeping the surfactant EXL concentration constant) or different concentrations of surfactant EXL (while keeping the dioxetane VPPD concentration constant) provide enhanced luminescence (as measured in RLU) compared to EXL and PPD. In addition, note that Figure 2B and 2C 6 μL of AP8 was used in Figure 2A 10 μL of AP8 was used in . The RLU observed with 10 μL of AP8 (not shown) was higher, less variable, and even more improved relative to EXL and PPD than with 6 μL of AP8.

[0237] Figure 3 Shown is a comparison of the luminescence observed for VPPD using EXL surfactant compared to VPPD, PPD, and LP530, normalized to the intensity of VPPD in EXL surfactant.

[0238] from Figure 1 , 2A As observed in Figures 2C and 3, VPPD exhibited higher RLUs in a relatively short time (e.g., about 4 minutes) relative to PPD and VMPD, regardless of the surfactant system (LP530 vs. EXL surfactant). Figure 3 The significant relative difference in brightness in RLU between VPPD in EXL compared to LP530 is highlighted. For example, the brightness of VPPD in EXL is more than 20 times higher than LP530 (about 9,500 RLU in EXL surfactant compared to about 380 in LP530). One benefit of the compositions described herein is that the detection limit of alkaline phosphatase can be improved. Using the compositions described herein, not only can ALP in AP10 (1.2×10 -21 mol / μL ALP), and ALP in AP11 and AP12 (1.2×10 -22 mol / μL and 1.2×10 -23 mol / μL ALP concentration).

[0239] Example 5

[0240] Dioxetane compositions containing the dioxetane concentrations, surfactant / enhancer concentrations, oxetane:surfactant ratios, and magnesium amounts described in Example 4 were prepared using the following surfactants / enhancers (1) to (4):

[0241]

[0242] *Note that Surfactant / Enhancer 2 is a mixture of 1,4- and 1,3-isomers.

[0243] Used to generate Figure 4 The oxetanes and enhancer combinations for the data are listed in Table 1 below.

[0244] Oxetane Enhancer A VPPD (1) B VPPD (1) C VPPD (2) D VPPD (3) E VPPD (4) F PPD Fluorescein

[0245] Figure 4 A comparison of the luminescence observed for VPPD using surfactant / enhancers (1) to (4) when mixed with AP8 is shown compared to LP530. Specifically, 100 μL of a solution containing VPPD and surfactant / enhancer was mixed with 10 μL of AP8 and then incubated at 37°C.

[0246] from Figure 4 It was observed that compared with LP530, Surfactants / enhancers (regardless of whether they are polymers or small molecules) Surfactant / enhancer) produced significantly higher RLUs in a relatively short time (e.g., about 4 minutes). Another observation is that small molecules Surfactants (2) and polymers Surfactant / enhancer (4) has about the same RLU at 4 minutes. And there appears to be a difference in the RLU at 4 minutes (and beyond) between heteropolymeric surfactant / enhancers (3) and (4), with (3) being brighter than (4).

Claims

1. A composition comprising a compound of formula I and a salt thereof, and at least one Surfactants: Where R 1 and R 2 Each is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon to which they are attached, they provide C5-C 10 Cycloalkyl ring; R 3 It is C1-C 10 Alkyl, C6-C 10 Aryl or heteroaryl; R 4 It is C2-C 10 alkenyl; R 5 Is H or C1-C 10 Alkyl; and X is a phosphate ester.

2. The composition of claim 1, wherein the composition comprises a surfactant.

3. The composition of claim 1, wherein the composition comprises two or more surfactants.

4. The composition of claim 1 or 3, wherein the Surfactants are selected from small molecules Surfactants and Polymerization Surfactant.

5. The composition of claim 1 or 4, wherein the Surfactants include polymers Surfactant, the polymer The surfactant comprises repeating units (A), repeating units (B), or both: Wherein "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl".

6. The composition of claim 5, wherein the molar ratio of the repeating unit (A) to the repeating unit (B) is from 1:100 to 100:

1.

7. The composition of claim 1 or 4, wherein the polymer Surfactants contain the following structure: wherein z is an integer from 2 to 1000, or from 100 to 600.

8. The composition of claim 1 or 4, wherein the polymer Surfactants include small molecules of the formula Surfactants: in: R 12 To R 14 Each is independently C1-C 10 alkyl; R 15 is arylalkyl; and X - It is a counter ion.

9. The composition of claim 8, wherein R 15 is an arylalkyl group substituted with a C2-C8 alkenyl group or a group of the formula: where k is 0, 1 or 2; R 16 To R 18 Each is independently C1-C 16 alkyl.

10. The composition of claim 9, wherein R 16 To R 18 Each is independently C4-C 10 alkyl.

11. The composition of claim 9, wherein the C2-C8 alkenyl group is a group of the formula: Where R 19 It is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl.

12. The composition of claim 1, wherein the composition further comprises a fluorescein surfactant.

13. The composition of claim 12, wherein the fluorescein surfactant has formula (V): Where R 6 It is C6-C 20 alkyl, and optionally comprises an ammonium salt, such as hexadecyltrimethylammonium bromide; and optionally, wherein the fluorescein surfactant is 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein or 5-stearoylaminofluorescein.

14. The composition of claim 1, wherein the phosphate group has the formula -OP(O)(ONa)(ONa).

15. The composition of claim 1, wherein the compound of formula I is a compound of formula II or a salt thereof: Where R 10 and R 11 are independently H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl.

16. The composition of claim 15, wherein R 10 and R 11 Each is independently H or halogen.

17. The composition of any one of claims 1 to 16, wherein the compound of formula I is a compound of formula III or a salt thereof:

18. The composition of any one of claims 1 to 16, wherein the compound of formula I is a compound of formula IV or a salt thereof:

19. The composition of any one of claims 1 to 16, wherein the compound of formula I is a compound of the following formula or a salt thereof:

20. The composition of any one of claims 1 to 16, wherein the compound of formula I is a compound of the following formula or a salt thereof:

21. The composition of any preceding claim further comprising an amine buffer.

22. A composition according to any preceding claim in the form of an aqueous composition.

23. The composition of any preceding claim, wherein the composition comprises alkaline phosphatase, and wherein the concentration of alkaline phosphatase in the composition is at most 1×10 -18 mol / μL、1×10 -19 mol / μL、1×10 -20 mol / μL or 1×10 -21 mol / μL.

24. The composition of claim 23, wherein the concentration of alkaline phosphatase in the composition is at least 1.2×10 -22 mol / μL or at least 1.2×10 -23 mol / μL.

25. The composition of claim 23 or 24, wherein the composition comprises at least 1×10 -22 or at least 1×10 - 21 mol ALP.

26. The composition of claim 23, 24 or 25, wherein the composition comprises at most 1×10 -17 mol ALP, up to 1×10 -18 mol ALP, up to 1×10 -19 mol ALP or at most 1×10 -20 mol ALP.

27. A method of detecting alkaline phosphatase in a sample comprising contacting the sample with a composition according to any preceding claim to produce a mixture, and monitoring luminescence of the mixture.

28. The method of claim 27, wherein the concentration of alkaline phosphatase in the mixture is at most 1×10 - 18 mol / μL, up to 1×10 -19 mol / μL, up to 1×10 -20 mol / μL or up to 1×10 -21 mol / μL.

29. The method of claim 27 or 28, wherein the concentration of alkaline phosphatase in the mixture is at least 1×10 -22 mol / μL or at least 1×10 -23 mol / μL.

30. The method of any one of claims 27, 28 or 29, wherein the mixture comprises at most 1×10 -17 molALP, up to 1×10 -18 molALP, up to 1×10 -19 molALP or up to 1×10 -20 mol ALP.

31. The method of any one of claims 27, 28, 29 or 30, wherein the mixture comprises at least 1×10 - 21 mol ALP or at least 1×10 -22 mol ALP.

32. The method of any one of claims 27, 28, 29, 30 or 31, further comprising measuring the intensity of the resulting luminescence and correlating the intensity to the presence of the alkaline phosphatase or the concentration of the alkaline phosphatase.

33. The method of any one of claims 27 to 32, wherein the luminescence has at least one of: a peak RLU greater than 8,000; and a T of 3 minutes or less. 1 / 2 .

34. The method of any one of claims 27 to 33, wherein the sample is an aqueous sample having a pH of 4 to 12.

35. The method of claim 27, wherein the sample is an aqueous sample having a pH of 8 to 12.

36. The method of any one of claims 27 to 35, wherein the method comprises contacting the sample with a The method comprises contacting the sample with a composition comprising PPD and a fluorescein surfactant having the formula: wherein p is an integer from 2 to 10, such as an integer from 5 to 9 or from 5 to 7.

37. The method of claim 36, wherein the improved signal-to-noise ratio is at least 50-fold.

38. The method of claim 36, wherein in the absence of the sample, the The surfactant composition has a luminescence RLU of less than 15.

39. The method of claim 36, wherein in the absence of the sample, the The RLU of luminescence of the surfactant composition is greater than 1 and less than 15.

40. A kit for determining the presence of alkaline phosphatase, the kit comprising a compound of formula I and a salt thereof, and at least one Surfactant, and optionally, wherein the composition comprises a surfactant, the formula I is: Where R 1 and R 2 Each is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon to which they are attached, they provide C5-C 10 Cycloalkyl ring; R 3 It is C1-C 10 Alkyl, C6-C 10 Aryl or heteroaryl; R 4 It is C2-C 10 alkenyl; R 5 Is H or C1-C 10 alkyl.

41. The kit of claim 40, wherein the Surfactants are selected from small molecules Surfactants and Polymerization Surfactant.

42. The kit of claim 40 or 41, wherein the polymer The surfactant comprises repeating units (A), repeating units (B), or both: Wherein "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl".

43. The kit of claim 42, wherein the molar ratio of repeating unit (A) to repeating unit (B) is 1:100 to 100:

1.

44. The kit of claim 40 or 41, wherein the polymer Surfactants contain the following structure: wherein z is an integer from 2 to 1000, or from 100 to 600.

45. The kit of claim 40 or 41, wherein the small molecule The surfactant has the formula: in: R 12 To R 14 Each is independently C1-C 10 alkyl; R 15 is optionally substituted arylalkyl; and X- is the counter ion.

46. ​​The kit of claim 45, wherein R 15 is an arylalkyl group substituted with a C2-C8 alkenyl group or a group of the formula: where k is 0, 1 or 2; R 16 To R 18 Each is independently C1-C 16 alkyl.

47. The kit of claim 46, wherein R 16 To R 18 Each is independently C4-C 10 alkyl.

48. The kit of claim 46, wherein the C2-C8 alkenyl group is a group of the formula: Where R 19 It is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl.

49. The kit of claim 40, wherein the kit further comprises a fluorescein surfactant, wherein the fluorescein surfactant has formula (V): Where R 6 It is C6-C 20 alkyl, and optionally comprises an ammonium salt, such as hexadecyltrimethylammonium bromide; and optionally, wherein the fluorescein surfactant is 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein or 5-stearoylaminofluorescein.

50. The kit of claim 49, wherein the surfactant is 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein, or 5-stearoylaminofluorescein.

51. The kit of any one of claims 40 to 50, wherein the compound of formula I and salts thereof, and at least one surfactant are in separate containers.

52. The kit of any one of claims 40 to 50, wherein the compound of formula I and salts thereof, and at least one surfactant are in a single container.

53. The composition, method or kit of any preceding claim, wherein the dioxetane concentration is between 100 mg / L and 150 mg / L.

54. The composition, method or kit of any preceding claim, wherein the surfactant concentration is 200 mg / L to 300 mg / L.

55. The composition, method or kit of any preceding claim, wherein the ratio of dioxetane to surfactant is from 1:1 to 1:

3.

56. The composition, method or kit of any preceding claim, wherein the composition comprises 50 mg / L to 500 mg / L of magnesium (II) salt.

57. The composition, method or kit of any preceding claim, wherein the composition comprises 50 mg / L to 500 mg / L MgCl2.

58. A method for detecting alkaline phosphatase in a sample, comprising: a. Provide a dioxetane compound and A mixture of surfactants; b. mixing the sample with the dioxetane compound and a mixture of surfactants to produce a mixture comprising the sample; and c. monitoring luminescence of a mixture comprising the sample, wherein monitoring the luminescence comprises detecting a peak RLU greater than 8,000 or a T of 3 minutes or less 1 / 2 .

59. The method of claim 58, wherein in the absence of the sample, the dioxetane compound and the The peak RLU of luminescence of the combination of surfactants is less than 15.

60. The method of claim 58, wherein in the absence of the sample, the dioxetane compound and the The peak RLU of luminescence of the combination of surfactants is greater than 1 and less than 15.

61. The method of any one of claims 58 to 60, wherein the concentration of alkaline phosphatase in the mixture comprising the sample is at most 1×10 -18 mol / μL、1×10 -19 mol / μL、1×10 -20 mol / μL or 1×10 -21 mol / μL.

62. The method of any one of claims 58 to 61, wherein the concentration of alkaline phosphatase in the mixture comprising the sample is at least 1×10 -22 mol / μL or 1×10 -23 mol / μL.

63. The method of any one of claims 58 to 62, wherein the mixture comprising the sample comprises at most 1×10 -17 mol, 1×10 -18 mol, 1×10 -19 mol or 1×10 -20 mol ALP.

64. The method of any one of claims 58 to 63, wherein the mixture comprises at least 1×10 -21 mol or 1×10 -22 mol ALP.

65. A method for detecting alkaline phosphatase in a sample, comprising: a. mixing the sample with a dioxetane compound and a first mixture of surfactants to produce a second mixture comprising the sample; and b. monitoring the luminescence of the second mixture, wherein monitoring the luminescence comprises detecting a peak RLU greater than 8,000 or a T of 3 minutes or less 1 / 2 .

66. The method of claim 65, wherein in the absence of the sample, the dioxetane compound and the The peak RLU of luminescence of the combination of surfactants is less than 15.

67. The method of claim 65, wherein in the absence of the sample, the dioxetane compound and the The peak RLU of luminescence of the combination of surfactants is greater than 1 and less than 15.

68. The method of any one of claims 65 to 67, wherein the concentration of alkaline phosphatase in the second mixture is at most 1×10 -18 mol / μL、1×10 -19 mol / μL、1×10 -20 mol / μL or 1×10 -21 mol / μL.

69. The method of any one of claims 65 to 68, wherein the concentration of alkaline phosphatase in the second mixture is at least 1×10 -22 mol / μL or 1×10 -23 mol / μL.

70. The method of any one of claims 65 to 69, wherein the second mixture comprises at most 1×10 -17 mol, 1×10 -18 mol, 1×10 -19 mol or 1×10 -20 mol ALP.

71. The method of any one of claims 65 to 70, wherein the second mixture comprises at least 1×10 -21 mol or 1×10 -22 mol ALP.

72. The method of any one of claims 65 to 70, wherein the second mixture comprises: At least 10 μL, at least 50 μL, at least 100 μL; and / or Up to 100 μL, Up to 110 μL, Up to 150 μL, Up to 200 μL, or Up to 500 μL.

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